Our goal is to determine the basis for HIV-1 integrase function through an understanding of the molecular structure of full-length integrase with and without its DNA substrate. We seek to use this knowledge to facilitate development of new anti-HIV therapeutics against a new target. Combination drug therapy that blocks multiple steps in HIV virus replication will almost certainly be the only way to continue to make progress against HIV infections. Resistance to currently available drugs against viral reverse transcriptase, and protease is an ever-increasing problem. HIV-1 integrase, the third viral enzyme, also essential for viral replication, is a most attractive, validated, target for new anti-HIV drug development. This proposal builds on our development of a soluble, crystallizable form of full-length HIV-1 integrase, our recently published 2.68, resolution structure of the integrase catalytic core and C-terminal domains, and our 1.68, resolution structure of the isolated catalytic core domain, in combination with our ongoing efforts to identify new small molecule inhibitors of integrase. Our first aim is to determine the arrangement of all the functional domains of full length integrase, initially tractable at low (-68,) resolution. Using this structure, and the high resolution structures of its domains as our guide, our second aim seeks progressively higher resolution structures by modifying contacts at the crystal lattice interface. Our third aim involves extensive attempts to generate co- crystals involving HIV-1 integrase and DNA constructs that mimic intermediates in the integration event. These three structural aims should advance significantly our understanding of the mechanism of integrase function and provide the ideal template for developing new integrase inhibitors. As such, our fourth aim is to harness structural and computational methods to develop inhibitors of integrase as drug leads. Inhibitor target sites include the enzyme active site and three non-active site locations. The latter include the dimer interface, the flexible linker connecting the core and C-terminal domains, and the viral DNA binding platform. Inhibitor co-crystallization with multiple crystal forms of the core, multidomain, and DNA-complexes will define inhibitor mechanisms of binding and action. Our fifth aim is to use in vitro and cell-based assays to test the biological hypotheses generated from our structural data and to test and validate inhibitor mechanisms of action.